Our goal is to attain an understanding of the mechanism of nucleocytoplasmic transport. This active governs the regulated flow of macromolecules and information between the nucleus and cytoplasm during normal cell growth. Nuclear import involves three distinct steps: First, specific Nuclear Localization Signals (NLS) in transport substrates appear to be recognized by specific receptors, which may be soluble. Second, transport substrates are bound in a docked configuration to the Nuclear Pore Complex (NPC) transporter. Finally, substrates (and possibly the receptor) are translocated across the nuclear envelope and released. This work will focus on three facets of transport in amphibian oocytes using cryoelectron microscopy, 3-dimensional image processing and X-ray crystallography: (a) The docking and translocation phases of transport. Recent work has shown that the central transport assembly within NPCs is trapped in at least 4 transport-related configurations in rapidly isolated nuclei. This data has resulted in the formulation of a double-iris model of the NPC- transporter. Separate 3-dimensional structures will be determined of NPCs with transporters in closed, docked, "in transit" and open configurations. This will result in a detailed understanding of the molecular mechanism of NPC-mediated nucleocytoplasmic transport. Furthermore, the positions of gp190/210 and a putative transport ATPase within the NPC will be mapped by cytochemical labeling and image processing of frozen-hydrated specimens. (b) Nucleoplasmin transport receptor recognition. The amphibian nuclear protein Nucleoplasmin (NP) is involved in the sequestration and assembly of histones H2A & H2B into nucleosomes. Nucleoplasmin will be purified from oocytes and over-expressed in E. coli for crystallization studies and for use as an affinity probe to purify the oocyte transport receptor. A Xenopus lambda-gt expression library will then be screened for transport- receptor cDNAs and suitable clones isolated, sequenced and over-expressed. Ultimately, high resolution crystal structures of the NP-pentamer and NP- receptor complexes should provide detailed insights into the function of the NLS in nuclear import.